The field of metabolic engineering has been successful in producing medically important natural products such as the anti-malarial drug artemisinin, which was previously very expensive. Development of high-titer production pathways for such compounds has been slow, however, largely due to the necessity of laborious screening of different pathway variants. I propose to overcome these challenges using new high-throughput evolutionary. Our long-term goal is to engineer new organisms that will contribute to solving the problems of adequate and timely supply of important pharmaceuticals. The rationale for this project is that more versatile and more high-throughput approaches are needed to facilitate the pursuit of this goal. The objective of this proposal is to demonstrate new evolutionary methods for developing biosensors and high-titer production of new medically important compounds. Accordingly, I propose the following specific aims: Specific Aim #1 Engineer Escherichia coli to produce high titers of the anticancer and anti- inflammatory terpenoid zerumbone. This will involve building and optimizing the functionality of three non-native genes. Specific Aim #2 Develop a positive-negative selection scheme for engineering the specificity of G-protein-coupled receptors. For positive selection, a gene required for growth will only be expressed when the receptor binds the compound of interest. For negative selection, a lethal gene will be expressed if the receptor is activated in the absence of the compound. Specific Aim #3 Develop a versatile platform for generating biosensing reporter microbes. Olfactory sensor proteins with engineered specificities expressed in one strain will detect production of the target compound by the other strain, and in response generate a reporter or a molecule required for growth by the producing strain. These studies are innovative in developing new approaches to synthesizing drugs and biosensors, and in applying synthetic ecosystems toward production of high-value products. Upon completion, I expect to have significantly expanded the tool-kit available for rapidly engineering receptors and for reaching high-level production of complex compounds.